A member of the flavivirus genus of the Flaviviridae family, WNV is a neurotropic enveloped virus with a single-stranded, positive-polarity 11-kilobase RNA genome and is closely related to viruses that cause dengue fever, yellow fever, and the Japanese, Saint Louis, and Murray Valley encephalitides. WNV cycles between mosquitoes and birds but also infects humans, horses, and other vertebrate species. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks throughout the United States during the past five years indicate that it has established its presence in the Western Hemisphere. Infected humans develop a febrile illness that can progress rapidly to a meningitis or fatal encephalitis syndrome. See Granwehr et al., 2004, Lancet Infect Dis 4:547-56; Hubalek et al., 1999, Emerg Inf Dis 5:643-650; and Petersen et al., 2003, JAMA 290:524-8. At present, treatment is supportive and no vaccine exists for human use.
The molecular and structural basis of antibody-mediated protection against WNV and other flaviviruses remains speculative. Based on the sequencing of in vitro neutralization escape variants and the site-specific substitution of specific charged or polar residues, most neutralizing antibodies against flaviviruses appear to localize to domain III. See Beasley and Aaskov, 2001, Virology 279:447-58; Beasley et al., 2002, J. Virol. 76:13097-13100; Cecilia and Gould, 1991, Virology 181:70-7; Crill and Roehrig, 2001, J. Virol. 75:7769-73; Lin et al., 1994, Virology 202:885-90; Roehrig et al., 1983, Virology 128:118-26; Schlesinger et al., 1996, J. Gen. Virol. 77:1277-85; Seifet al., 1995, Vaccine 13:1515-21; Volk et al., 2004, J. Biol. Chem. 279:38755-38761; and Wu et al., 1997, Virus. Res. 51:173-81. However, putative contact residues for individual mAbs that have been identified by neutralization escape may be flawed because mutations may cause local unfolding that abolish multiple antibody epitopes. Moreover, many of these studies did not confirm that the mapped neutralizing mAbs also abolished infection in vivo in animals. As an alternative strategy, one group recently used NMR to map a neutralizing mAb against Japanese encephalitis virus (JEV). See Wu et al., 2003, J. Biol. Chem. 278:46007-46013. Chemical shifts in domain III of JEV were detected after mAb binding at residues E302-312, E322-329, E360-372, and E385-392, corresponding to the top portion of an exposed β-barrel. Although additional information was obtained, NMR and its solution structure do not provide any insight as to the structural basis for antibody recognition of the neutralizing epitope.
Recently, prophylactic and therapeutic efficacy of pooled, immune human γ-globulin has been demonstrated in mice infected with WNV. See Agrawal and Petersen, 2003, J. Infect. Dis. 188:1-4; Ben-Nathan et al., 2003, J. Infect. Dis. 188:5-12; and Engle et al., 2003, J. Virol. 77:12941-12949. Because human γ-globulin is made from human blood plasma, it has an inherent risk of transmitting known and unknown infectious agents. More recently, a monoclonal antibody (mAb) therapeutic against WNV E protein has been developed that is ˜1,000-fold more potent that pooled human γ-globulin in its ability to neutralize virus infection in vitro and in vivo. This antibody (E16), which recognizes domain III, was cloned, humanized, expressed and confirmed as therapeutically effective in an established mouse model of WNV infection.
Nonetheless, additional antibodies that can bind and neutralize WNV more strongly may be needed for effective treatment and/or prevention of WNV infection. Such antibodies could be designed and constructed by identifying amino acids of the antibodies that mediate the antigen-antibody interaction. These amino acids can be selectively altered to generate antibody variants that could be screened for enhanced WNV binding and/or neutralization.
Further, small molecule therapeutics that can mimic antibodies that bind and/or neutralize WNV infection would also be of use, as such small molecules may be easier and less expensive to manufacture and easier to administer orally. Such small molecule therapeutics could be designed based on the three-dimensional structure coordinates of an antibody that binds domain III of WNV E protein in complex with the domain III.
In addition, a small molecule therapeutic such as an antigen that mimics the domain III epitope recognized by E16 could be administered to generate an immune response against WNV. A composition comprising an antigen that mimics WNV would provide a safer method of preventing WNV infection. An effective antigen mimic of WNV could be administered, to persons with a functioning immune system, as an immunoprophylactic to raise an immune response against the virus with minimal or no danger of infection caused by the immunoprophylactic itself.
Still further, a small molecule therapeutic that can interact with antibody-WNV complexes to stimulate antibody-mediated neutralization of WNV infection would also be of significant utility in treatment and/or prevention of WNV infection. In addition, a greater understanding of the interaction between neutralizing antibodies and domain III of WNV E protein is needed to inform strategies for designing vaccines that will elicit strong and broadly neutralizing immune responses. The present invention provides a substantial advancement towards these and other unrealized needs.